This application represents a multidisciplinary effort to investigate the mechanisms for lung defense against oxidative stress. The hypothesis for the program is that generation of reactive oxygen species (ROS) in patients at risk plays a critical role in the initiation of lung damage leading to a cascade of events that culminates in the syndrome of acute lung injury (ALl). The major focus will be on antioxidant enzymatic protection with special focus on a newly described lung antioxidant enzyme, peroxiredoxin 6 (Prdx 6). Results generated during a period of R-01 support to study this enzyme indicate that Prdx 6 can reduce phospholipid hydroperoxides which provides a basis for protection against oxidant stress through reversal of membrane lipid peroxidation. We will utilize mouse models of altered Prdx 6 expression to determine the role of this enzyme in antioxidant defense, will evaluate control of Prdx 6 expression under oxidant stress, and will characterize the biochemical requirements for enzymatic activity with respect to binding and catalysis of phospholipid hydroperoxides. We will identify functional polymorphisms in specific antioxidant genes that are associated with an increased risk of ALl among patients with major trauma. The functional significance of the polymorphisms will be determined by analysis of the promoter region, if relevant, or expression of the mutant protein. We will employ physical, mass spectrometric, and proteomic techniques to characterize oligomedzation and oxidative modification of Prdx 6 as well as to determine the effects of Prdx 6 expression levels on oxidative modification of other lung proteins dudng oxidative stress. We will also search for changes in blood proteins levels that are diagnostic of oxidative stress and development of ALl. We will investigate a novel strategy for anti-oxidant defense through use of polymer nanocarriers for delivery of antioxidant enzymes to endothelium. A major emphasis will be on the cytosolic delivery of Prdx 6. We will be supported by: 1) clinical, biostatistical and data management; 2) molecular engineering and protein preparation; and 3) cell culture and animal husbandry. This represents both basic science and translational studies that will provide new insights into antioxidant defense and potential mechanistic-based therapies for ROS-mediated ALl.